Identification and Validation of Cyclic Peptides with Mucin-Selective, Location-Specific Binding in the Gastrointestinal Tract

Abstract

Oral drug delivery is a widely preferred method of drug administration due to its ease of use and convenience for patients. Localization of drug release in the gastrointestinal (GI) tract is important to treat localized diseases and maximize drug absorption. However, achieving drug localization in the dynamic GI tract is challenging. To address this challenge, we leveraged the geographic diversity of the GI tract by targeting its mucus layers, which coat the epithelial surfaces. These layers, composed of mucin glycoproteins, are synthesized with unique chemical compositions and expressed in different regions, making them ideal targets for drug localization. In this article, we identify cyclic peptides that bind selectively to MUC2 (in the intestines) and MUC5AC (in the stomach), serving as targeting ligands to these regions of the GI tract. We demonstrate the effectiveness of these peptides through in vitro, ex vivo, and in vivo experiments, showing that incorporating these targeting ligands can increase binding and selectivity 2-fold to the desired regions, thus potentially overcoming challenges with localizing drug distribution in oral delivery. These results indicate that cyclic peptides can be used to localize drug cargoes at certain sites in the body compared to free drugs.

Keywords: binding; gastrointestinal; localization; mucin; peptide; specificity.

Figure 1

Schematic of one type of stomach-specific and intestine-specific drug delivery approach (peptide-conjugated nanoparticles) based on binding to the mucin glycoproteins found in these environments (MUC5AC in the stomach, MUC2 in the intestines). In this scenario, drug-loaded particles contain cyclic peptides conjugated to the surface, which confer targeting capability. The cyclic peptides are conjugated to the surface of the nanoparticle as shown in the figure, where the dark blue circles represent cysteine amino acids, and the other circles represent the amino acids that compose the cyclic peptide. The legend at the bottom shows which elements of the structure correspond to different regions of the mucins (D-domain, cysteine-rich region, PTS domain, and cysteine knot). Schematic of mucin structures adapted from Subramanian et al.

Figure 2

In vitro identification and validation of mucin-binding peptides. Peptides were identified from a large library of cyclic peptides using phage display (a), and peptides were identified for MUC2 (b) and MUC5AC (c); the sequences shown reflect the middle seven amino acids (X₇) within the CX₇C peptide structure to reflect the common sequences and motifs. To validate their performance, phage ELISA was employed (d), and the background-subtracted phage ELISA results were obtained for MUC2-binding hits (e) and MUC5AC-binding hits (f). In (b) and (c), red boxes represent common sequences, while blue boxes represent common motifs (or sequences of three or more amino acids that are shared among two distinct peptide sequences). In (e) and (f), statistical significance is conveyed by the following: *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (one-way ANOVA and post-hoc Bonferroni for multiple comparisons).

Figure 3

Ex vivo binding analysis for identified hits. The fluorescently tagged peptides were incubated with magnetically clamped pieces of mucin-containing tissue, and the fluorescence of the bound nanoparticles was semiquantitatively analyzed using in vivo imaging and spectroscopy (a). Representative IVIS images for measurement of the ex vivo binding of MUC2-selective peptides to the small intestine (b) and stomach (c) and MUC5AC-selective peptides to the small intestine (d) and stomach (e) are shown here. “Score” results indicating the average ex vivo binding magnitude are shown for the MUC2-binding (f) and MUC5AC-binding (g) hits to the tissue of interest (small intestine for MUC2-selective peptides and stomach for MUC5AC-selective peptides); the score is calculated by normalizing the individual fluorescence values against those of the negative and positive controls for each plate. Overall selectivity to the mucin of interest is shown for the MUC2-binding (h) and MUC5AC-binding (i) hits. The top hits are shown for each mucin with black boxes based on their binding strength and selectivity.

Figure 4

In vivo analysis of top predicted peptide hits. Representative IVIS images are shown for M2.3, along with the free AF647 and WGA-AF647 controls and blank PBS (a). The fluorescence intensity in each organ (esophagus, stomach, and small intestine) is shown for M2.3 and the controls (b); the results shown subtracted the tissue background and the fluorescence values from the blank group rats. Similar IVIS images (c) and tissue fluorescence results (d) are also shown for M5.2, free AF647, and WGA-AF647. Finally, the overall selectivity is shown for M2.3 in (e) and M5.2 in (f), each compared to the controls. Upper values in fluorescence scale bars in (a) and (c) are defined as follows: (a) 3 × 10⁹, (c) 1.2 × 10⁹. Statistical significance is designated by *p < 0.05, ***p < 0.005, ****p < 0.001 (two-tailed Student’s t-test for single comparisons, one-way ANOVA and post-hoc Bonferroni for multiple comparisons). n = 6 for in vivo experiments.